Draft genome sequences of 11 Xanthomonas strains associated with bacterial spot disease in Turkey

Bacterial spot is an economically significant disease in tomato and pepper-producing countries globally. We report the whole-genome sequence of 11 Xanthomonas strains associated with bacterial spot disease on pepper, tomato and eggplant in the Southeastern Anatolia Region, Turkey. This genomic information can be used as a reference to study the genetic diversity of these species and contribute to illuminating pathogen evolution with respect to host specificity.


INTRODUCTION
Bacterial spot is considered a major disease in many regions worldwide where tomatoes and peppers are grown, causing a significant economic impact to growers. Bacterial spot disease is caused by four Xanthomonas spp.: X. euvesicatoria, X. perforans, X. vesicatoria, and X. hortorum pv. gardneri [1][2][3]. While the severity of the disease can vary depending on the specific bacterial strain [4], and environmental factors [5], yield losses of up to more than 40 % on peppers and severe effects on the yield and quality of tomatoes have been reported [6,7]. These bacterial pathogens are capable of being disseminated through the movement of seed, infected transplants, contaminated tools, wind-blown rain and dust particles, and insect vectors among others [3]. The disease has been identified in more than 70 countries across all continents except Antarctica [8]. Pepper and tomatoes represent the two major hosts for these pathogens. Prior to 2010, X. perforans was only known to cause bacterial spot of tomato. However, in the last decade, a host range shift in the X. perforans population to include pepper as a host has been observed in North America [2,3,9,10]. Surveys of Xanthomonas populations in other growing regions are needed to identify host-range shifts. There is currently no genomic information available for Xanthomonas on pepper and tomato from Turkey, where both crops are the most widely grown vegetables. In 2021, Turkey reported 3 million tons of pepper and 13 million tons of tomato production and was ranked as third and fourth producer, respectively, in the world (FAOSTAT, 2021). Hence, it is crucial to understand the diseases that can cause millions of dollars in losses impacting the economy of the country.

Sample collection and isolation of bacteria
Symptomatic leaves showing symptoms as small, dark-brown lesions surrounded by yellow halo were collected from three different solanaceous plants: pepper, tomato and eggplant from Southeastern Anatolia Region of Turkey in 2020. A small section from the margin of individual lesions was macerated in 25 µl of sterile tap water using sterilized scalpels and forceps. A loopful of the suspension was streaked on nutrient agar (NA) medium (Laboratories, Detroit, MI) in a quadrant streak pattern, and plates were incubated for 72 h at 28 °C until single colonies were observed. An individual yellow colony was streaked on fresh NA and, growth from the 24 h culture was suspended in cryovials containing 30 % glycerol in nutrient broth and stored at −80 °C for further assays.

Pathogen identification and pathogenicity test
Overnight fresh cultures grown on NA were transferred to nutrient broth and incubated overnight at 28 °C by placing on a shaker at 250 r.p.m. Genomic DNA was extracted from overnight nutrient broth cultures using the Gram-negative bacterial DNA extraction protocol from the Wizard Genomic DNA Purification Kit (Promega, Madison, WI). Initial species confirmation was conducted using species-specific real-time PCR probes to amplify the hrcN gene [11]. As explained by [11], each real-time PCR reaction consisted of 0.2 µl of molecular-grade water, 5 µl of 10×PCR buffer, 6.0 µl of 25 nM MgCl 2 , 0.6 µl of 10 mM dNTP, 3.0 µl of each forward and reverse primer at 2 µM, 1.25 µl of each probe at 1 µM, and 0.2 µl of platinum Taq per and 2 µl extracted genomic DNA. A sample with a cycle threshold (Ct) value <35 was considered as positive whereas a sample with a Ct value ≥37 was considered as negative [11]. X. euvesicatoria strain E3 and X. perforans strain 91-118 were used as controls.
Pepper (Capsicum annuum L.) cultivar 'Early California Wonder' (ECW), and tomato cultivar 'Bonny Best' were infiltrated in the underside of leaves with bacterial suspensions adjusted to 10 8 c.f.u. ml −1 (OD 600 =0.3) from overnight cultures grown on NA. Infiltrated leaf areas were monitored for a hypersensitive response (HR) or susceptible reaction. Three strains, X. perforans Xp-91-118 (pathogenic on tomato), X. perforans Xp-2010 (pathogenic on pepper) and X. euvesicatoria Xe_E3 (pathogenic on both tomato and pepper), were used as controls. Because eggplant has not been reported as a host for X. perforans, in-planta bacterial growth was assessed for the Tu_04 strain by infiltrating a bacterial suspension adjusted to 10 5 c.f.u. ml −1 into two cultivars of eggplant (Hybrid and Black beauty). Pathogenicity test was conducted once with each test strain and control strain in single leaf while population growth assay was replicated two times.

Genome sequencing
For whole-genome sequencing, extracted genomic DNA (as explained above) was sent to Microbial Genome Sequencing Center (MIGS; Pittsburgh, PA, USA). Genomes were sequenced using the Illumina Next-Seq2000 platform, generating 151 bp pairedend reads. Sample libraries were prepared using the Illumina DNA Prep kit and IDT 10 bp UDI indices. Demultiplexing, quality control and adapter trimming was performed with bcl-convert (v3.9.3) -a proprietary Illumina software for the conversion of bcl files to basecalls.

Genome assembly
Raw reads were assembled using pipelines published in Timilsina et al. [12]. Briefly, adapters were eliminated by Trim Galore with default settings to generate clean sequences and pair raw reads [13]. Paired reads were assembled using SPAdes (v.3.10.1) with '-careful' switch [14] and contigs with less than 500 bp and k-mer coverage of less than 2.0 were eliminated. Validated reads were aligned against the filtered contigs using default parameters of Bowtie 2 (v. 2.3.3) [15]. The generated SAM files were converted to BAM files using SAMtools [16]. Pilon was used to polish the draft genome assemblies with '--frags' setting [17]. Genome statistics were calculated using a python script (https://github.com/sujan8765/nepgorkhey_python/blob/master/genome_stats.py). Pyani (v.0.2.10) [18] was used to calculate an average nucleotide identity (ANI) among assemblies and to reference genomes of type strains of X. euvesicatoria LMG-27970 and X. perforans DSM-18975. Genome assemblies were annotated using the Prokaryotic Genome Annotation Pipeline v.6.4 from the National Centre for Biotechnology Information [19].

RESULTS AND DISCUSSION
Of 11 strains, five from pepper were identified as X. euvesicatoria, and six strains from tomato, pepper and eggplant as X. perforans (Table 1). Pathogenicity tests revealed that all strains from tomato and pepper were pathogenic on their host of isolation. X. perforans strain Tu_04 showed weak growth in two different eggplant cultivars, reaching populations of only 1.6×10 7 c.f.u. cm -2 ( Fig. 1). Bacterial spot is not typically reported on eggplant, but in this case, the diseased plants in the field were surrounded by tomato (Fig. 2).
A de novo assembly of the genomes of 11 Xanthomonas strains generated assemblies with the number of contigs ranging from 34 to 147 (average = 60) and sequencing coverage ranging from 84 to 102 (average =94). The genome length varied from 4.91 to 5.18 Mbp with an average of 5.07 Mbp. Genomes had an average N 50 of 291 926 bp. Whole-genome ANI analysis was consistent with initial identifications by real-time PCR. The five X. euvesicatoria strains had >99.8 % and 98.5 % ANI with type strain of X. euvesicatoria (i.e. LMG_27970) and the type strain of X. perforans (i.e. DSM_18975), respectively. The six X. perforans strains showed >99.8 % and 98.6 % ANI with the X. perforans type strain and X. euvesicatoria type strain, respectively. The NCBI annotation for all X. perforans strains from Turkey predicted 91 RNA genes and 53 tRNA genes except for Tu_04 strain, which has 89 RNA genes and 51 tRNA genes, whereas all X. euvesicatoria strains from Turkey contain 95 RNA genes and 52 tRNA genes. The predicted number of protein coding genes varies from 4050 to 4312 (average =4199) in all 11 strains. Genome statistics of all sequenced strains, NCBI annotation data along with the isolated host are reported in Table 1 and pairwise ANI is presented in Table 2.
X. perforans, formerly known as bacterial spot pathogen on tomato only, has recently been shown to have expanded its host range to pepper as an emerging pathogen [2,3,9,10]. We found X. perforans to be highly pathogenic on pepper and weakly pathogenic on eggplant, supportive of the host expansion. This could have resulted either from gene flow from Xanthomonas species infecting pepper     [2] or mutations in the genes that affect pathogenicity on pepper [20]. The genetic factors, however, involved in host specificity in X. perforans remain poorly understood. Future research investigating the extent of host expansion and adaptation is needed to precisely characterize host-pathogen interaction and understand the associated mechanisms contributing to the successful host expansion of X. perforans.

Funding information
This work was supported in part by the United States Department of Agriculture Specialty Crops Research Initiative project grant no. 2019-51181-30010 from the USDA National Institute of Food and Agriculture.

Author contributions
All authors contributed equally to conceptualization, analysis of the results and writing of the manuscript.

Conflicts of interest
The authors declare that there are no conflicts of interest.
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Peer review history * As the authors have used an in-house python script to calculate assembly statistics, could they please make this script available?
Re: Revised as suggested. We uploaded the script in github and cited the URL in revised version. * As NCBI have annotated the genomes, could the authors please comment on these annotations (e.g., number of genes, number of rRNAs, number of tRNAs)?
Re: Revised as suggested. We have added the information in results section as well as in Table 1. However, we could not find the NCBI annotation for Type strain (LMG_27970) so, we left blank in Table 1 for this particular strain only.
* Do the authors have data for eggplant infections from their other isolates to compare to isolate Tu-4?
No, we do not have any data from other strains on eggplant. Since, eggplant was not previously reported as a host for bacterial spot associated Xanthomonas spp.We do not have any other strains isolated or reported to be pathogenic on eggplant except Tu-4. * Could the authors please conduct a phylogenetic analysis of their isolates. Although their ANI results confirm that they are all Xanthomonas (and, with an ANI>95%, suggest that X. perforans and X. euvesicatoria are a single species), it does not place them within their evolutionary context.
Thank you for your careful review and taking the time to identify the factors that could strengthen the manuscript. We really appreciate the suggestion for the additional analysis, however our main objective for this manuscript is "Genome Announcement" to make availability of genomes from Turkey for future comparative study. We will address the evolutionary studies including phylogenetic analysis of these strains in reference to other global strains in future manuscripts.

* For the infection assays, could the authors please report the protocol used to infect leaves in sufficient detail for it to be replicated from infection to enumeration? Could the authors also report the number of infections carried out, and if these infections were carried out on separate leaves or one leaf? Did the authors carry out a control on a tomato or pepper plant to demonstrate the isolates were infectious?
Re: Revised as suggested under Method Section; subsection Pathogen Identification and Pathogenicity test.

* For the RT-PCR, could the authors please briefly explain the assay and the cutoff values used to assign species identifications?
Re: Revised as suggested under Method Section; subsection Pathogen Identification and Pathogenicity test.RT-PCR assays was performed based on previous work from Strayer et al. 2016 without any modification. That is why we cited that reference. However, for the ease of reading we briefly explained in revised version as per reviewer suggestion.

Presentation of results
The results are presented satisfactorily, however there are minor changes needed: * Table 1: Could the authors please change Genome Length (Mb) to Genome Length (Mbp). Could the authors also include the same statistics for the reference genomes used in this work?
Re: Revised as suggested

* Table 2: Could the authors please provide an ANI-scaled coloured background to the cells to make the ANI values easier to parse? Alternatively, the results could be presented as a heatmap.
Re: Revised as suggested * Figure 1: As there is not an even length of time between measurements, could the authors please present this data as a bar graph instead of a line graph, or space the x-axis evenly? In addition, the y axis is mislabelled as "Log10/cm2" where it presumably should be Log10CFU/cm2.
The population growth graphs are preferably illustrated as line graph, so we decided to keep as line graph instead of bar graph. However, as per reviewer suggestion we evenly spaced out the X-axis. We corrected the y-axis label.

* Figure 2: Do the researchers have a photo of the leaf used in panel C at day 0 of infection and if so, could they please include this?
Re: Revised as suggested. We have included the Day 0 picture as per the suggestion.

How the style and organization of the paper communicates and represents key findings
The submission could benefit from a clearer subdivision into Introduction, Methodology, Results, and Discussion sections. The authors describe the work they have carried out in a logical order.
Re: Revised as suggested. Our main focused of this manuscript was genome announcement, so we wanted to keep it as short as possible. However, we followed the reviewer suggestion and divided in subheadings as Introduction, Methods, Results and Discussion.

Literature analysis or discussion
The authors sufficiently review the literature to familiarise the readership with Xanthomonas and its importance as an agricultural pathogen. Could the authors please expand on the significance of their finding of eggplant colonising Xanthomonas and on the phylogeny and taxonomy of Xanthomonas as a genus and how their isolates fit into it? As their ANI calculations suggest that all of their isolates are monospecific, could they also please discuss how their isolates compare to other published Xanthomonas populations? Finally, could the authors provide citations for Trim Galore and SPAdes.
We understand the reviewer's perspective; however, we decided not to expand the mentioned information in this manuscript. As again, we want to keep concise as a genome announcement only and perform further in-depth analyses in future manuscript. Since X. perforanswas only weakly pathogenic on eggplant, we do not believe we need to expand this discussion. As such we do not want to make a big concern out of it in this manuscript.
Citation -Revised as suggested.

Any other relevant comments
I believe there are substantial changes required to the manuscript's content in order to ensure reproducibility of the analyses they have carried out. In addition, I believe that the authors need to substantially expand the methodology descriptions for how they cultured and isolated their strains and carried out infection assays. The additional analyses I have suggested do not preclude this work from publication, however are not onerous and would improve the value of the work carried out.
Thank you for your careful review and taking the time to identify the factors that could strengthen the manuscript. We really appreciate the suggestion and try our best to incorporate all the comments in revised version. We have expanded and explained in detail about methodology as per the suggestions. However, for some additional analysis, specifically phylogenetic analysis we decided to perform in future.

Reviewer 2 Comments to Author: Review -Subedi et al. Access Microbiology
* The authors performed Real-time PCR amplification to check the identity of the isolated strains. They claim that these PCR amplifications were performed using species-specific probes. However, the authors did not mention the gene markers they amplified. It will be helpful to add the information regarding the amplified genes and the primers used for the PCR analysis.
Re: Revised as suggested under Method Section; subsection Pathogen Identification and Pathogenicity test.Thank you for this observation. We have included the target gene (hrcN).RT-PCR assays was performed based on previous work from Strayer et al. 2016 without any modification. We have briefly explained the method in the revised version. Furthermore, we want to concise the manuscript as short as possible So, we want to refer the paper for detail explanation.
* The authors performed whole genome sequencing (WGS) on the 11 isolated Xanthomonas strains. Here information about the sequencing procedure is missing such as the library preparation procedure and sequencing depth used per library.
Thank you for this comment. We sent the samples for sequencing to a company and received the raw data and summary report with few information. We added information from the report briefly in revised version.

* Finally, can the authors discuss the mechanism behind Xanthomonas host range shift or speculate how this does work and if any genomic region transfer, or acquisition triggers adaptation to new hosts?
We understand the reviewer's perspective; however, we do not know about the underlying mechanism for host range shift, nor can we speculate without any preliminary analysis. At this point, we do not want to conjecture regarding mechanism behind host shift. However, we have added a paragraph under discussion section about the speculations from other studies. Xanthommonas is a bacterial pathogen that is reported to cause spot disease, which results in a loss in yields of globally essential crops. With this study, the authors by performing WGS, could provide a resource for genomic information extracted from 11 Xanthomonas strain, that can be used to study genetic diversity and strainspecific markers that would contribute to the understanding of Xanthomonas pathogenicity and host specificity, which are valuable to develop strategies to eradicate this pathogen. The data presented in this paper is generally scientifically fine. The manuscript is relatively easy to follow. However, we list some points below that we feel should be addressed before publication. Relating to experimental procedure : * The authors performed Real-time PCR amplification to check the identity of the isolated strains. They claim that these PCR amplifications were performed using species-specific probes. However, the authors did not mention the gene markers they amplified. It will be helpful to add the information regarding the amplified genes and the primers used for the PCR analysis. * The authors performed whole genome sequencing (WGS) on the 11 isolated Xanthomonas strains. Here information about the sequencing procedure is missing such as the library preparation procedure and sequencing depth used per library. * Finally, can the authors discuss the mechanism behind Xanthomonas host range shift or speculate how this does work and if any genomic region transfer, or acquisition triggers adaptation to new hosts? Relating to Annoncement : -(Lines 39-41): this sentence needs rephrasing.

Please rate the manuscript for methodological rigour Good
Please rate the quality of the presentation and structure of the manuscript

Very good
To what extent are the conclusions supported by the data? Strongly support Comments: 1. Methodological rigour, reproducibility and availability of underlying data The authors have provided sufficient information to access both raw sequencing reads and assemblies from NCBI. Likewise, the methods they have employed to assemble and analyse their genomes are sound. However, could the authors please address the following concerns: * C o u l d the authors please report culture conditions (Media, temperature, shaking speeds) used to grow cultures up for both DNA extraction and for leaf infection assays? * Could the authors please provide the protocol used to isolate these strains as well as a description of the symptoms used to identify bacterial spot disease? * Could the authors please report the settings used when executing Trim Galore, SPAdes, pilon, and pyani? * As the authors have used an in-house python script to calculate assembly statistics, could they please make this script available? * As NCBI have annotated the genomes, could the authors please comment on these annotations (e.g. number of genes, number of rRNAs, number of tRNAs)? * Do the authors have data for eggplant infections from their other other isolates to compare to isolate Tu-4? * Could the authors please conduct a phylogenetic analysis of their isolates. Although their ANI results confirm that they are all Xanthomonas (and, with an ANI>95%, suggest that X. perforans and X. euvesicatoria are a single species), it does not place them within their evolutionary context. * For the infection assays, could the authors please report the protocol used to infect leaves in sufficient detail for it to be replicated from infection to enumeration? Could the authors also report the number of infections carried out, and if these infections were carried out on separate leaves or one leaf? Did the authors carry out a control on a tomato or pepper plant to demonstrate the isolates were infectious? * For the RT-PCR, could the authors please briefly explain the assay and the cutoff values used to assign species identifications? 2. Presentation of results The results are presented satisfactorily, however there are minor changes needed: * Table 1: Could the authors please change Genome Length (Mb) to Genome Length (Mbp). Could the authors also include the same statistics for the reference genomes used in this work? * Table 2: Could the authors please provide an ANI-scaled coloured background to the cells to make the ANI values easier to parse? Alternatively, the results could be presented as a heatmap. * Figure 1: As there is not an even length of time between measurements, could the authors please present this data as a bar graph instead of a line graph, or space the x-axis evenly? In addition, the y axis is mislabelled as "Log10/cm2" where it presumably should be Log10CFU/cm2. * Figure 2: Do the researchers have a photo of the leaf used in panel C at day 0 of infection and if so could they please include this? 3. How the style and organization of the paper communicates and represents key findings The submission could benefit from a clearer subdivision into Introduction, Methodology, Results, and Discussion sections. The authors describe the work they have carried out in a logical order. 4. Literature analysis or discussion The authors sufficiently review the literature to familiarise the readership with Xanthomonas and its importance as an agricultural pathogen. Could the authors please expand on the significance of their finding of eggplant colonising Xanthomonas and on the phylogeny and taxonomy of Xanthomonas as a genus and how their isolates fit into it? As their ANI calculations suggest that all of their isolates are monospecific, could they also please discuss how their isolates compare to other published Xanthomonas populations? Finally, could the authors provide citations for Trim Galore and SPAdes. 5. Any other relevant comments I believe there are substantial changes required to the manuscript's content in order to ensure reproducibility of the analyses they have carried out. In addition, I believe that the authors need to substantially expand the methodology descriptions for how they cultured and isolated their strains and carried out infection assays. The additional analyses I have suggested do not preclude this work from publication, however are not onerous and would improve the value of the work carried out.
Please rate the manuscript for methodological rigour Poor